The current invention relates to a class of microbial coding sequences that are specifically induced during infection of a host by a microbial pathogen and more particularly to a set of probes that may be used to identify and isolate microbial virulence genes. The products of these virulence genes will provide potential targets for the development of vaccines or antimicrobial agents.
Microbial pathogens, or disease-producing microorganisms, can infect a host by one of several mechanisms. For example, they may enter through a break in the skin, they may be introduced by vector transmission, or they may interact with a mucosal surface. Disease ensues following infection of a host, when the potential of the pathogen to disrupt normal bodily functions is fully expressed. Each disease-producing microorganism possesses a collection of virulence factors, that enhance their pathogenicity and allow them to invade host or human tissues and disrupt normal bodily functions. Infectious diseases have been major killers over the last several thousand years, and while vaccines and antimicrobial agents have played an important role in the dramatic decrease in the incidence of infectious diseases, infectious diseases are still the number one cause of death world-wide.
Attempts to vaccinate are almost as old as man""s attempt to rid himself of disease. However, during the last 200 years, since the time Edward Jenner deliberately and systematically inoculated a population with cowpox to avoid a smallpox epidemic, vaccination, at least in parts of the world, has controlled the following nine major diseases: smallpox, diphtheria, tetanus, yellow fever, pertussis, poliomyelitis, measles, mumps and rubella. In the case of smallpox, the disease has been totally eradicated. The impact of vaccination on the health of the world""s population is hard to exaggerate. With the exception of safer water, no other modality, not even antibiotics, has had such a major effect on mortality reduction and population growth.
Following the first exposure of a host to an antigen, the immune response is often slow to yield antibody and the amount of antibody produced is small, i.e., the primary response. Upon secondary challenge with the same antigen the response is more rapid and of greater magnitude, i.e., the secondary response. Achieving an immune state equal to the accelerated secondary response following reinfection with a pathogenic microorganism is the goal that is sought to be induced by vaccines. Vaccines are basically suspensions of viral, bacterial, or other pathogenic agents or their antigens which can be administered prophylactically to induce immunity.
In general, active vaccines can be divided into two general classes: subunit vaccines and whole organism vaccines. Subunit vaccines are prepared from components of the whole organism and are usually developed in order to avoid the use of live organisms that may cause disease, or to avoid the toxic components present in whole organism vaccines.
The use of purified capsular polysaccharide material of H. influenza type b as a vaccine against the meningitis caused by this organism in humans is an example of a vaccine based upon an antigenic component. See Parks et al., J. Inf. Dis., 136 (Suppl.):551 (1977), Anderson et al., J. Inf. Dis., 136 (Suppl.):563 (1977); and Mxc3xa4kela et al., J. Inf. Dis., 136 (Suppl.):543 (1977). Classically, subunit vaccines have been prepared by chemical inactivation of partially purified toxins, and hence have been called toxoids. Formaldehyde or glutaraldehyde have been the chemicals of choice to detoxify bacterial toxins. Both diphtheria and tetanus toxins have been successfully inactivated with formaldehyde resulting in a safe and effective toxoid vaccine which has been used for over 40 years to control diphtheria and tetanus. See, Pappenheimer, A. M., Diphtheria. In: Bacterial Vaccines (R. Germanier, ed.), Academic Press, Orlando, Fla., pp. 1-36 (1984); Bizzini, B., Tetanus. Id. at 37-68. In contrast to subunit vaccines, whole organism vaccines make use of the entire organism for vaccination. The organism may be used killed or alive (usually attenuated) depending upon the requirements necessary to elicit protective immunity. The following discussion will focus on live but attenuated microorganisms (live vaccines).
In the case of intracellular pathogens, it is generally agreed that live vaccines induce a highly effective type of immune response. Ideally, these attenuated microorganisms maintain the full integrity of cell-surface constituents necessary for specific antibody induction yet are unable to cause disease, because they fail to produce virulence factors, grow too slowly, or do not grow at all in the host. Additionally, these attenuated strains should have no probability of reverting to a virulent wild-type strain. Traditionally, live vaccines have been obtained by either isolating an antigenically related virus from another species, by selecting attenuation through passage and adaptation in a nontargeted species or in tissue cultures, or by selection of temperature-sensitive variants.
In contrast to these somewhat haphazard approaches of selecting for live vaccines, modern developmental approaches introduce specific mutations into the genome of the pathogen which affect the ability of that pathogen to induce disease, that is, specific mutations are introduced into genes involved in virulence. Defined genetic manipulation is the current approach being taken in an attempt to develop live vaccines for various diseases caused by pathogenic microorganisms. U.S. Pat. No. 5,210,035, exemplifies this approach by describing the construction of vaccine strains from pathogenic microorganisms made non-virulent by the introduction of complete and non-reverting mutational blocks in the biosynthesis pathways, causing a requirement for metabolites not available in host tissues. Specifically, Stocker teaches that S. typhi may be attenuated by interrupting the pathway for biosynthesis of aromatic (aro) metabolites which renders Salmonella auxotrophic (i.e., nutritionally dependent) for p-aminobenzoic acid (PABA) and 2,3-dihydroxybenzoate, substances not available to bacteria in mammalian tissue. These aroxe2x88x92 mutants are unable to synthesize chorismic acid (a precursor of the aromatic compounds PABA and 2,3-dihydroxybenzoate), and no other pathways in Salmonella exist that can overcome this deficiency. As a consequence of this auxotrophy, the aroxe2x88x92 deleted bacteria are not capable of extensive proliferation within the host; however, they reside and grow intracellularly long enough to stimulate protective immune responses.
Unfortunately the development of vaccines based on chemical toxoids, discussed previously, is difficult since protective antigens and the genes encoding them must first be identified and then procedures must be developed to efficiently isolate the antigens. Similarly, modem approaches to the rational development of live vaccines has been hampered by the limited knowledge available concerning genes that are involved in virulence and thus the targets of mutagenesis.
The medical literature up to about 1930 is full of vivid descriptions of gruesome infections by streptococci, staphylococci, and clostridia. The dawning of the age of antimicrobial therapy, with the introduction of the sulfonamides in the 1930s, allowed physicians finally to cure many of these fatal infections. From the outset, antibiotics were heralded as a panacea for everything from fungus-infected pear orchards to the common cold. Penicillin lozenges were popular as were nostrums such as antibiotic mouthwashes and throat sprays. By the 1950s, doctors jubilantly predicted an end to infectious diseases and, by the 1980s, half of all drug companies had stopped developing antibiotics, believing the battle won.
The stunning success of the pharmaceutical industry in the United Sates, Japan, the United Kingdom, France, and Germany in creating new antibiotics over the past three decades have caused society to become complacent about the potential of bacterial resistance, but what once was a situation where antibiotic controls prevailed has since deteriorated badly. C. T. Walsh, in a technical paper entitled xe2x80x9cVancomycin Resistance: Decoding the Molecular Logic,xe2x80x9d Science, 261:308-309 (1993) stated that xe2x80x9c[t]he 1990s may come to be remembered as a decade in which infectious diseases made a dramatic worldwide resurgence, largely because of the appearance of antibiotic-resistant microbes.xe2x80x9d
In economic terms alone, such antibiotic resistance is costly. A recent estimate is that the extra expense of treating multiresistant infections is $100 to $200 million annually in the United States, see A. Gibbons, Science, 257:1036-1038 (1992). But economic impact reflects only part of the true costs of dealing with antibiotic resistant infections. More than 13,000 Americans are dying each year from drug resistant bacteria and doctors warn that the problem is steadily worsening. The FDA considers bacterial drug resistance threatening enough that it is planning incentives to encourage development of new antibiotics.
To date, the vast majority of antibiotics in the marketplace were derived from large-scale screens or from analog development programs. Classification of antibiotics by mechanisms of action appears below in Table 1.
As is shown in Table 1, there are very few mechanisms of action that are exploited by current antibiotics. Unfortunately, to date the majority of antimicrobial agents have been randomly discovered. Robotic systems can perform thousands of tests per day by means of radioactive labeling or spectroscopic detection making it feasible to scan 100,000 to 500,000 compounds in a year. While the efforts are still in their early stages, some companies are beginning to use. xe2x80x9crational drug designxe2x80x9d to design new drugs that can use selective mechanisms to destroy a specific microbe. Understanding the biological or biochemical mechanism of a disease often suggests the types of molecules needed for new drugs. Consequently, not knowing what makes infectious diseases virulent in the first place, is a fundamental fact which has severely limited the continued development of vaccines and antibiotics. A method of identifying genes that are expressed by microbial pathogens infecting a host has been developed: in vivo expression technology (IVET).
Essentially, the IVET selection strategy disclosed in U.S. Pat. No. 5,434,065, and herein incorporated by reference originates with a microbial strain carrying a mutation in a biosynthetic gene that highly attenuates its growth in a given host. Next, growth of the mutant strain in the host is complemented by transcriptional fusions to the same biosynthetic gene. Although, in theory, many different biosynthetic genes (e.g., aroA, thyA, asd) could be used in this selection scheme, initial efforts have focused on the purA gene of Salmonella typhimurium, purA mutants are highly attenuated in their ability to cause mouse typhoid and to persist in host tissues. This purA requirement provides a basis for the positive selection of microbial virulence genes that are specifically induced in a given host.
The first step in construction of purA operon fusions as per U.S. Pat. No. 5,434,065 was to build a pool of recombinant clones containing random fragments of Salmonella DNA. Partial Sau3A I restriction digests of total S. typhimurium DNA were used to obtain the random DNA fragments, which were then cloned 5xe2x80x2 to an artificial operon having a promoterless purA gene fused to a promoterless lacZY gene on the vector, pIVET1. In the recombinant plasmids of interest, the fragment contained a Salmonella promoter in the proper orientation to drive the purA-lac fusion. This random pool was then introduced into a purA deletion strain of S. typhimurium that does not contain the Pi replication protein. Selection for ampicillin resistance requires the integration of the recombinant plasmids into the chromosome by homologous recombination, using the cloned Salmonella DNA as the source of homology. In the clones of interest, the product of the integration event generates a duplication of Salmonella material in which one promoter drives the purA-lac fusion, while the other promoter drives the expression of a wild-type copy of the putative virulence gene as shown in FIG. 1. The expression of both of these promoters is selected in the host. Expression of the purA-lac fusion is selected to overcome the parental purA auxotrophy. Expression of the virulence gene is selected because the gene product is required for infection. The expression levels of the operon fusions can be monitored both on laboratory media and in animal tissues by measuring the levels of xcex2-galactosidase activity.
A large collection of recombinant plasmids that contained the purA-lac transcriptional fusions were integrated into the chromosome of a purA deletion strain of S. typhimurium, FIG. 1. The subsequent pool of integrated fusion strains was injected intraperitoneally (i.p.) into a BALB/c mouse. After a 3 day incubation, the mouse was sacrificed and the bacteria were recovered from an internal organ such as the spleen, intestine, or liver. Only those bacterial cells that contain fusions to chromosomal promoters that had sufficient transcription levels to provide enough of the purA gene product were selected (to overcome the parental purine deficiency) by demanding the survival and propagation of the fusion strain in the host. Note that all genes that have constitutively active promoters will answer the IVET selection because they would produce sufficient levels of purA gene product (and LacZ) all the time. Thus, when the mouse-selected pool was plated on MacConkey Lactose indicator medium, an increase in the percentage of Lac+ clones is expected compared to the pre-selected pool. This expected shift has been termed the xe2x80x9cRED SHIFT.xe2x80x9d To test the prediction, the percentage of Lac+ clones in the pre-selected and mouse-selected fusions was determined by plating on MacConkey Lactose indicator medium. In the pre-selected pool, 50% of the fusions were transcriptionally active or xe2x80x9cONxe2x80x9d in vitro (red or pink in colonies), whereas in the mouse-selected pool 95% of the fusions were xe2x80x9cON.xe2x80x9d This observed shift in percentage in favor of Lac+ clones (the RED SHIFT) suggests that the IVET system selected for promoters that are active in vivo. Since the underlying premise of IVET is that some virulence genes will be expressed only when they are in the proper environment and not on simple laboratory media, we focused our efforts on the rare 5% Lacxe2x88x92 class of fusions that were recovered from the spleens of infected mice. Presumably, these Lace strains contained fusions to genes that were xe2x80x9cONxe2x80x9d in the mouse (to complement the purA deficiency) and xe2x80x9cOFFxe2x80x9d out of the mouse.
While the IVET approach provides an important new way to identify genes that are involved in virulence, some shortcomings were encountered using the IVET method discussed above. There is still a need, therefore, for a method and a means for identifying and isolating microbial virulence genes the products of which will provide a basis for rational vaccine and drug design.
Accordingly, it is an object of this invention to identify a class of microbial virulence genes involved in virulence.
It is an additional object of this invention to enhance the selectivity of methods currently available to identify virulence genes.
It is a further object of this invention to provide a set of coding sequences known to be involved in pathogenesis for use as probes to identify and isolate other microbial genes that are cotranscribed with said coding sequences during infection.
Additional objects, advantages and novel features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities, combinations, compositions, and methods particularly pointed out in the appended claims.
To achieve the foregoing and other objects and in accordance with the purposes of the present invention, as embodied and broadly described therein, the method and compositions of this invention comprise using a class of coding sequences to identify genes, the transcription or cotranscription of which are induced during microbial infection of a host.